1. Field of the Invention
The present invention relates, in general, to antioxidant responsive elements (AREs). In particular, the present invention relates to a DNA construct comprising an ARE having the DNA sequence 5'-RGR AC NNN GCT-3' (SEQ ID NO: 1) operably linked to a heterologous protein coding sequence; cells and non-human organisms comprising the DNA construct; a method of screening for a compound that increases transcription of an mRNA regulated by an antioxidant responsive element; and a purified compound (for example, a protein) that binds to an antioxidant responsive element.
2. Related Art
Epidemiological studies have demonstrated that lowering low density lipoprotein-cholesterol (LDL-C) or raising high density lipoprotein-cholesterol (HDL-C) reduces cardiovascular risk (Waters D., and Lesperance, Am. J. Med. 91(1B):10S-17S (1991)). However, among myocardial infarction survivors, greater than one half have normal lipid levels, suggesting that factors other than lipoprotein profiles contribute to the disease process (Wissler, R.W., Am. J. Med. 91(1B):3S-9S (1991)). One such factor appears likely to be the oxidation of LDL (Steinberg, D., et al., N. Engl. J. Med. 320:915-924 (1989); Parthasarathy, S., and Rankin, S. M., Prog. in Lipid Res. 31:127-143 (1992); Esterbauer, H., et al., Free Radical Res. Commun. 6:67-75 (1989)). Oxidized LDL has been implicated in the formation of foam cells and thus may play an important role in the etiology of atherosclerosis (Sparrow, C. P., et al., J. Biol. Chem. 264:2599-2604 (1989); Ross, R., N. Engl. J. Med. 314:488-500 (1986)). In contrast, oxidized HDL is not avidly taken up by macrophages, does not lead to foam cell formation (Parthasarathy, S., et al., Biochim. Biophys. Acta. 1044:275-283 (1990)) and may actually inhibit endothelial cell-mediated LDL modification (Parthasarathy, S., et al., Biochim. Biophys. Acta. 1044:275-283 (1990); van Hinsbergh, V. W., et al., Biochim. et Biophys. Acta. 878:49-64 (1986)). HDL is also capable of protecting against LDL peroxidation in vitro (Parthasarathy, S., et al., Biochim. Biophys. Acta. 1044:275-283 (1990); Klimov, A. N., et al., Biokhimiia 54:118-123 (1989); Mackness, M. I., et al., FEBS Lett. 286:152-154 (1991)). The antioxidative activity of HDL has been demonstrated in vivo (Klimov, A. N., et al., Atherosclerosis 100:13-18 (1993)). These properties suggest another protective role for HDL (in addition to its involvement in `reverse cholesterol transport`) in reducing atherosclerotic risk.
Reduced levels of plasma HDL are observed in cigarette smokers (Haffner, S. M., et al., Arteriosclerosis 5:169-177 (1985); Assmann, G., et al., J. Clin. Chem. & Clin. Biochem. 22:397-402 (1984)). However, the mechanisms responsible for the decrease are not known. During cigarette smoking, the oxidation of polycyclic aromatic hydrocarbons produces free radicals (Pryor, W. A., et al., Environ. Health Perspect. 47:345-355 (1983)). The presence of quinone and hydroquinone complexes in the particulate phase of cigarette smoke can result in generation of reactive species such as superoxides and hydrogen peroxide. If a metal catalyst is present, hydroxyl radicals will also form. Consequently, the smoker has a higher free radical burden and a lower HDL level than the nonsmoker and it has been suggested that this may contribute to the smoker's higher risk of developing atherosclerosis (Wilhelmsson, C., et al., Lancet 1:415-420 (1975)).
The major protein component of HDL is apolipoprotein (apo) AI, which is believed to promote the process of "reverse cholesterol transport" (Gotto et al., Methods Enzymol. 128: 3-41 (1986); Miller et al., Nature (London) 314: 109-111 (1985); Glomset, Adv. Intern. Med. 25: 91-116 (1980)). In this process, excess cholesterol is liberated from the peripheral tissues and carried, via HDL, to the liver for degradation. In addition, apo AI acts as a cofactor for the enzyme lecithin-cholesterol acyltransferase (LCAT), which is also involved in reverse cholesterol transport (Gotto et al., Methods Enzymol. 128: 3-41 (1986); Miller et al., Nature (London) 314: 109-111 (1985); Glomset, Adv. Intern. Med. 25: 91-116 (1980)). Further evidence that apo AI is a strong negative factor for atherosclerosis comes from experiments in which transgenic mice carrying the human apo AI gene were fed a high fat diet. Here, expression of the apo Al transgene and the resulting high levels of human apo AI in the animals' blood appeared to protect against development of fatty streak lesions (Rubin et al., Nature (London) 353: 265-267 (1991)).
The human apo AI gene is located on the long arm of chromosome 11. The DNA sequence of this gene is identified in Karathanasis et al., Nature (London) 304: 371-373 (1983); Breslow et al., Proc. Nat. Acad. Sci. USA 79: 6861-6865 (1982); and GenBank accession no. M20656. Cis- and trans-acting elements involved in the regulation of transcription of the apo AI gene have been studied by several groups (Sastry et al., Mol. Cell. Biol. 8. 605-614 (1988); Widom et al., Mol. Cell. Biol. 11: 677-687 (1991); Papazafiri et al., J. Biol. Chem. 266: 5790-5797 (1991); Pagani et al., J. Lipid Res. 31: 1371-1377 (1990); Smith et al., J. Clin. Invest. 89. 1796-1800 (1992); Sigurdsson et al., Arteriosclerosis and Thrombosis 12: 1017-1022 (1992); Tuteja et al., FEBS Letters 304: 98-101 (1992); Jeenah et al., Mol. Biol. Med. 7: 233-241(1990); and Tam et al., Canadian Patent Application No. 2,159,532, filed on Sep. 29, 1995 and laid open for public inspection on Mar. 30, 1997).
A consensus antioxidant responsive element (ARE) with the sequence 5'-RGTGACNNNGC-3' (SEQ ID NO: 33) is present in the rat glutathione S-transferase (GST) Ya subunit gene and the rat NAD(P)H:quinone reductase genes (Rushmore et al., J. Biol. Chem. 266:4556-4561 (1991)). Similarly, Li and Jaiswal (J. Biol. Chem. 267:15097-15104 (1992)) found within the human NAD(P)H:quinone oxidoreductase gene a sequence corresponding to the ARE described by Rushmore et al., supra.
Although a great deal of work has been done to date on the regulation of expression of the human apo AI gene and on antioxidant responsive elements in other genes, the mechanisms by which various antioxidants influence apo AI expression are heretofore unknown. Given the protection that high plasma apo AI levels provide, it would be extremely desirable to understand how a particular compound could increase apo AI expression. Additionally, novel AREs and convenient methods for screening for compounds which increase transcription of an mRNA regulated by an ARE would also be extremely desirable.